The present invention is directed to a polynucleotide comprising open reading frames defining a coding region encoding a retinoblastoma binding protein (RBP-7) as well as regulatory regions located both at the 5xe2x80x2end and the 3xe2x80x2end of said coding region. The present invention also pertains to a polynucleotide carrying the natural regulation signals of the RBP-7 gene which is useful in order to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region. The invention also concerns polypeptides encoded by the coding region of the RBP-7 gene. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention includes genetic markers, namely biallelic markers, that are means that may be useful for the diagnosis of diseases related to an alteration in the regulation or in the coding regions of the RBP-7 gene and for the prognosis/diagnosis of and eventual treatment with therapeutic agents, especially agents acting on pathologies involving abnormal cell proliferation and/or abnormal cell differentiation.
Among the genetic alterations that have been shown to represent direct or indirect causative agents of proliferative diseases, such as cancers, there may be cited mutations occurring at loci harboring genes that are called tumor suppressor genes.
Tumor suppressor genes are defined as genes involved in the control of abnormal cell proliferation and whose loss or inactivation is associated with the development of malignancy. Tumor suppressor genes encompass ortho-genes, emerogenes, flatogenes, and onco-suppressor genes.
More specifically, tumor suppressor genes are genes whose products inhibit cell growth. Mutant alleles in cancer cells have lost their normal function, and act in the cell in a recessive way in that both copies of the gene must be inactivated in order to change the cell phenotype. The tumor phenotype can be rescued by the wild-type allele, as shown by cell fusion experiments first described by Harris and colleagues (Harris H. et al., 1969). Germline mutations of tumor suppressor genes may be transmitted and thus studied in both constitutional and tumor DNA from familial or sporadic cases. The current family of tumor suppressors include DNA-binding transcription factors (i.e. p53, WT1), transcription regulators (i.e., RB, APC) and protein kinase inhibitors (i.e. p16).
The existence of tumor suppressor genes has been particularly shown in cases of hereditary cancers. These are cancer where there is a clear pattern of inheritance, usually autosomal dominant, with a tendency for earlier age of onset than for sporadic tumors.
Tumor suppressor genes are detected in the form of inactivating mutations that are tumorigenic. The two best characterized genes of this class code for the proteins RB (Retinoblastoma protein) and p53.
Retinoblastoma is a human childhood disease, involving a tumor in the retina. It occurs both as an inheritable trait and sporadically (by somatic mutation). Retinoblastoma arises when both copies of the RB gene are inactivated. In the inherited form of the disease, one parental chromosome carries an alteration in this region, usually a deletion. A somatic event in retinal cells that causes the loss of the other copy of the RB gene causes a tumor. Forty percent of cases are hereditary, transmitted as an autosomal dominant trait with 90% penetrance. Of these cases, around 10-15% are transmitted from an affected parent, the remaining arising as de novo germ-lime mutations. In the sporadic form of the disease, the parental chromosomes are normal, and both RB alleles are lost by somatic events. The tumor suppressor nature of RB was shown by the introduction of a single copy of RB1 into tumor cell lines lacking the gene, resulting in complete or partial suppression of the tumorigenic phenotype.
The RB protein has a regulatory role in cell proliferation, acting via transcription factors to prevent the transcriptional activation of a variety of genes, the products of which are required for the onset of DNA synthesis, the S phase of the cell cycle.
When investigating on the molecular function of RB, it has been found that the RB protein interacts with a variety of viral proteins, including several tumor antigens, such as SV40 T antigen, adenovirus E1A protein, human papillomavirus E7. These viral proteins have been shown to bind to RB, thereby inactivating it and allowing cell division to occur.
Thus, an important step toward defining a mechanism underlying tumor suppressor activity of the RB gene was the observation that the transforming products of adenovirus (E1A protein), simian virus 40 (large T antigen) and human papillomavirus (E7 protein) could precipitate wild-type RB protein. This, in turn, led to the identification of a family of cellular proteins that can reversibly bind to a discrete domain on the RB protein, referred to as the T/E1A pocket by using the same specificity as the viral products. The subsequent observation that protein binding was inhibited following RB protein phosphorylation in the late G, phase of the cell cycle suggested the hypothesis that the RB protein, as well as the related product p107, may regulate the functional activity of its binding partners by a cell-cycle dependent pattern of physical association. In particular, the activity of the RB protein has been shown to be regulated through cell cycle-dependent phosphorylation by cyclin-dependent kinases.
The picture of transcription regulation is made even more complex by the finding that a number of RB related proteins (e.g. p107 and p130) also bind members of the E2F family and are therefore involved in regulatory process.
In view of the foregoing, there clearly exists a pressing need to identify and characterize the cellular proteins that interact with the retinoblastoma protein in order to provide diagnostic and therapeutic tools useful to prevent and cure cell differentiation disorders, particularly disorders in which a lack of completion of cell differentiation, particularly in terminal cell differentiation, or in which an abnormal cell proliferation is detected, such as in proliferative diseases like cancer.
For the purpose of the present invention, cells with abnormal proliferation include, but are not limited to, cells characteristic of the following disease states: thyroid hyperplasia, psoriasis, benign prostatic hypertrophy, cancers including breast cancer, sarcomas and other neoplasms, bladder cancer, colon cancer, lung cancer, prostate cancer, various leukemias and lymphomas.
This invention is based on the discovery of a nucleic acid molecule encoding a novel protein, more particularly a retinoblastoma binding protein (RBP-7).
The present invention pertains to nucleic acid molecules comprising the genomic sequence of the gene encoding RBP-7. The RBP-7 genomic sequence comprises regulatory sequence located upstream (5xe2x80x2-end) and downstream (3xe2x80x2-end) of the transcribed portion of said gene, these regulatory sequences being also part of the invention.
The invention also deals with the complete cDNA sequence encoding the RBP-7 protein, as well as with the corresponding translation product.
Oligonucleotide probes or primers hybridizing specifically with a RBP-7 genomic or cDNA sequence are also part of the present invention, as well as DNA amplification and detection methods using said primers and probes.
A further aspect of the invention is recombinant vectors comprising any of the nucleic acid sequences described above, and in particular of recombinant vectors comprising a RBP-7 regulatory sequence or a sequence encoding a RBP-7 protein, as well as of cell hosts and transgenic non human animals comprising said nucleic acid sequences or recombinant vectors.
Finally, the invention is directed to methods for the screening of substances or molecules that inhibit the expression of RBP-7, as well as with methods for the screening of substances or molecules that interact with a RBP-7 polypeptide or that modulate the activity of a RBP-7 polypeptide.
The invention also concerns biallelic markers of the RBP-7 gene which can be useful for genetic studies, for diagnosis of diseases related to an alteration in the regulation or in the coding regions of the RBP- 7 gene and for the prognosis/diagnosis of an eventual treatment with therapeutic agents, especially agents acting on pathologies involving abnormal cell proliferation and/or abnormal cell differentiation